【作者】 张伟；

【导师】 李明；

【作者基本信息】 第二军医大学 ， 外科学， 2011， 博士

【摘要】 【背景】周围神经损伤(peripheral nerve injury, PNI)是临床最常见的创伤之一。随着现代建筑业、交通运输业的发展以及局部战争的频发,PNI的发生率也逐年呈上升趋势。由于成熟的神经元不能分裂和复制,与其它组织相比,周围神经损伤后的再生和恢复效果还很不理想。若不能及时救治,可导致肌肉功能丧失、感觉功能损害、功能恢复不佳甚至导致病人终生残疾,给社会带来巨大的损失和沉重的负担,已成为世纪医学挑战之一。目前国内外修复周围神经损伤的主要策略是桥接神经断端,促进神经轴突再生,克服再生屏障。20世纪90年代Lundborg利用神经再生室模型证实神经趋化特异性以来,神经导管修复神经缺损的优势逐渐被人们认识和接受。随着组织工程技术的飞速发展,周围神经损伤的修复又取得了新的进展。组织工程技术治疗PNI的基本模式是―种子细胞+细胞因子+生物支架‖。利用具有良好的组织相容性和生物活性的组织工程化人工神经搭载神经干细胞修复PNI取得了一定得疗效。【目的】1、体外培养骨髓间充质干细胞(BMSCs),并在体外诱导分化为骨髓源性神经干细胞,实现短时间内获得增殖能力较强的神经干细胞的要求。2、以PLGA为原料制作管壁具有三维结构的中空的可降解组织工程用神经导管。3、利用IKVAV自组装多肽构建组织工程化人工神经。4、将可降解神经导管、IKVAV自组装多肽凝胶、骨髓源性神经干细胞和神经生长因子构建的组织工程化人工神经移植至坐骨神经缺损处,观察神经再生和功能恢复情况,探讨新型组织工程化人工神经修复周围神经的可行性。【方法】1、利用全骨髓培养法进行原代骨髓间充质干细胞的培养。以3月龄大白兔为取材对象,于胫骨平台下抽取2~3ml骨髓,加入Percoll液中离心,10%FBS,1%抗生素的DMEM培养液重悬后,接种于培养皿中培养。BMSCs达到亚融合后,去除培养液,加入诱导液(DMEM培养基、全反式视黄酸、bFGF等),诱导BMSCs向类施万氏细胞分化。2、以PLGA为原料,通过静电纺丝技术制作中空的、管壁具有三维结构的可降解神经导管,并进行神经导管的体外、体内生物相容性测试。3、以IKVAV自组装多肽为原料,在一定条件下触发自组装形成多肽凝胶,并进行其与神经干细胞的相容性初步测试。4、将构建的组织工程化人工神经移植进行神经缺损修复实验。以新西兰大白兔为动物模型,实验动物随机分为3组:A组:自体神经移植组,B组:神经导管+神经干细胞+NGF,C组:神经导管+IKVAV自组装凝胶+神经干细胞+NGF。于术后3、6、9、12周,应用肌电图、肌肉湿重测量、HE染色、免疫荧光染色、透射电镜等观察方法观察神经干细胞的存活、周围神经功能的恢复情况。【结果】1、流式细胞仪鉴定结果表明成功分离、培养BMSCs,诱导后经S-100免疫细胞化学染色鉴定为类SC细胞,细胞纯度达87%。2、利用新型的静电纺丝工艺能够制作出管壁具有三维结构的组织工程用神经导管,导管外径3mm,内径2.5mm,管壁纤维直径约18μm,呈螺旋上升结构。导管管壁孔隙率约85.4%,支架具有良好的生物相容性,降解时间约为3个月。3、在实验室条件下成功触发IKVAV多肽自组装成凝胶,透射电镜显示其纤维直径为10-30nm,长度可达数百纳米,纳米纤维交织成立体网状结构。测定神经干细胞在凝胶上的黏附率及生物活性显示其有良好的生物相容性。4、构建的人工神经修复长节段坐骨神经缺损,术后3组动物均出现不同程度的足底溃疡,恢复情况以A组最好,C组次之,B组最差。神经肌电图、小腿三头肌湿重结果提示C组神经修复效果接近A组(P > 0. 05)而优于B组(P < 0. 05)。HE染色观察12周时A组见较多束状组织,神经纤维排列整齐,神经纤维较为粗大,髓鞘较厚。B、C组切片可见PLGA纤维基本降解、消失。C组亦可见较多束状的组织,呈波浪形,神经纤维较A组稀疏,组织间可见新生毛细血管,胶原组织较少。而B组中神经纤维排列较杂乱,胶原组织较多,神经纤维细小。透视电子显微镜观察:A组中有大量的有髓神经纤维,排列均匀。B组有髓神经纤维形态不规则,数量较少,少数神经纤维髓鞘轻度肿胀,呈脱髓鞘改变。C组中再生有髓神经纤维较多,但分布不均匀,纤维直径和髓鞘厚度大小不等,神经纤维间有较多新生血管形成。12周时组织工程化人工神经切片在荧光显微镜下观察到在神经损伤处有GFP荧光表达,表明神经干细胞在人工神经内仍然存活。【结论】1、采用全骨髓培养法进行BMSCs,可以获得大量高纯度BMSCs,经诱导后可以转化为类SC细胞,可作为神经修复的种子细胞。2、利用静电纺丝技术制作的PLGA可降解神经导管,具有良好的生物相容性和适当的降解时间,可以用来修复PNI。3、IKVAV多肽可以自组装形成凝胶,凝胶是由纳米纤维交联形成的类似细胞外基质的物质,具有良好的生物相容性和生物活性,可以作为神经组织工程支架。4、组织工程化人工神经修复坐骨神经具有较好的疗效,植入体内的神经干细胞可存活3个月以上,神经缺损处发现新生轴突,坐骨神经功能得到部分恢复。更多还原

【Abstract】 【Background】Peripheral nerve injury (PNI) is a frequently encountered trauma in clinic of orthopedic department. The incidence of PNI has been increase annually with the developing of modern architecture and transportation industry and the frequent local war. Compared with orther tissues, the prognosis of peripheral nerve injury is ugly because of limitied regenerative ability of mature neurons. Defuctionalization of muscle and sensory may occur without prompt remedy, and even permanent disability can happen to the patients, which brings about tremendous loss and burden. So the treatment of PNI has been the century challenge of medicine.Bridging the nerve gap, promoting regeneration of axon and overcoming regeneration barrier have been the main treatment of PNI. The advantage of nerve conduit in nerve injury treatment has been recognized and accepted since specificity of neurotropism was confirmed by Lundborg in 90s. Another progress has been made with the flying develop of tissue engineering technique, whose fundamental mode is "seed cell + cytokine + bioscaffold", and certain curative effect has been obtained with neural stem cell carried on tissue engineered artificial nerve which has outstanding histocompatibility and bioactivity.【Objectives】1、Bone marrow mesenchymal stem cells (BMSCs) is cultured in vitro and induced to differentiate into bone marrow-derived neural stem cells to obtain neural stem cells of higher proliferative capacity in a short period of time.2、Using PLGA as raw materials to produce a new type of three-dimensional structure biodegradable nerve conduit for tissue engineering.3、Using IKVAV self-assembled peptide to constructed tissue engineered artificial nerves.4、Tissue engineered artificial nerves constructed of biodegradable nerve conduit, IKVAV self-assembled peptide, bone marrow-derived neural stem cells and NGF were grafted to sciatic nerve defects to observe nerve regeneration and functional recovery, and to explore the feasibility of peripheral nerve repair using new type tissue-engineered artificial nerve. 【Method】1、We introduced a whole bone marrow culture method to culture primary BMSCs. Bone marrow(2~3ml) was obtained from 3-month-old rabbits under the tibial plateau, and was centrifugated in Percoll solution. The material was cultured after being re-floated in DMEM (10% FBS, 1% antibiotic). The culture fluid was removed after sub-fusion of BMSCs. Then induce fluid (DMEM, all-trans retinoic acid, bFGF) was added in to the material to induce the BMSCs differentiate toward Schwann cell-like.2、Degradable 3-D nerve conduit was structured by electrospun technique as PLGA being raw material, and biocompatibility was tested in vivo and in vitro.3、IKVAV self-assembling peptide was triggered into polypeptide-gel in certain condition, and compatibility of nerve stem cell was primarily tested.4、Nerve-repair experiment was performed by graft of tissue engineered artificial nerves. Animal models (New Zealand rabbits) were randomly divided into 3 groups: A: Auto-nerve transplatation group, B: Nerve conduct + NSC + NGF group, C: Nerve conduct + IKVAV + NSC + NGF group. The survival of NSCs and function recovery of PN was observed by means of EMG, muscle wet weight, HE stain, IMF stain and TEM at the time of 3, 6, 9, 12 weeks after operation.【Results】1、Flow cytometer indentification shows that BMSCs were isolated and cultured successfully. After the induction, BMSCs displayed morphologies of Schwann cell and its purity reached 87% by S-100 immunocytochemistry staining.2、The new type nerve conduit was produced by way of electrospun technique. We prepared new type of conduits whose outer diameter was 3mm, inner diameter was 2.5mm and the tube wall fiber diameters were 18μm. The fibers of the tube wall showed a spiral increasing arrangement in axial sections. The porosity of nerve conduit was 85.4%, and the 3D scaffold possessed features with good biocompatibi1ity, its degradation time was 3 months.3、We successfully trigger IKVAV peptides self-assemble into gels under laboratory conditions. The diameter of gels fibers was 10-30 nm and the length can reach to hundreds of nanometers. Nanofibers intertwined into three-dimensional mesh structure. Adhesion rate and biological activity determination showed that NSCs have good biocompatibi1ity.4、Different degree plantar ulcer were observed in all three groups (Severity: Group A> C> B) after reparation of long segment of sciatic nerve by artificial nerve. The result of Group C is significantly better than that of Group B (p<0.05) while it has no significant difference with that of Group A (p<0.05) in EMG test and determination of wet weight of triceps surae. More fasciculate tissue, thicker nerve fiber and neural sheath were observed in Group A but degradation of PLGA fiber in group B and C by HE stain after 12 weeks reservation. The nerve fiber were tangle and tiny with lots of collagen tissue in Group B, but raritas and neat with little collagen tissue in Group A, while much wave-shaped fascicularis tissue can be observed in Group C. TEM illustrated large quantity of neat medullated nerve fibers in Group A. Group B showed less quantity of irregular-shaped medullated nerve fibers and the medullary sheath of nerve fiber mild swelling under TEM. More regenerate medullated nerve fiber can be found in group C and more newborn capillary formation compared to group B under TEM. Fluorescence microscope illustrated that GFP fluorescence was expressed after 12 weeks, which showed that 12 weeks after transplantation, the NSCs were still alive over that period.【Conclusions】1、Using whole bone marrow culture method can get a large number of high-purity BMSCs and can be used as seed cells for tissue engineering technique for PNI repair after induction into Schwann cell-like.2、The PLGA biodegradable nerve conduits were suitable for PNI repair by tissue engineering technique with good biocompatibi1ity and biodegradability.3、IKVAV peptides can self-assembled into gels, which intertwined by nanofibers like extracellular matrix. The gels with good biocompatibility and biological activity and can be used as tissue engineering scaffold.4、Tissue-engineered artificial nerves can gain good effect for sciatic nerve injury repair. NSCs maintained a good biological activity living for 3 months. Newly born axon can be founded in the sciatic nerve defect. The function of sacistic nerve got partially restored.更多还原